CN117777262B

CN117777262B - Application of wheat TAALDHASE gene in regulation and control of wheat stem basal rot resistance

Info

Publication number: CN117777262B
Application number: CN202410199205.0A
Authority: CN
Inventors: 徐兆师; 马有志; 徐幸; 于太飞; 侯泽豪; 陈明; 陈隽; 周永斌; 王春宵
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2024-02-23
Filing date: 2024-02-23
Publication date: 2024-06-11
Anticipated expiration: 2044-02-23
Also published as: CN117777262A

Abstract

The invention discloses an application of wheat TAALDHASE gene in regulating and controlling wheat stem basal rot resistance. The invention provides an application of TAALDHASE protein in regulating and controlling wheat stem basal rot resistance; the TAALDHASE protein is any one of the following: (A1) a protein having the amino acid sequence of SEQ ID No. 1; (A2) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in (A1); the regulation is as follows: the expression level of TAALDHASE protein in wheat is reduced, and the resistance of the wheat to the stem rot is improved. The invention has important significance and application value for cultivating new varieties of wheat with stem rot resistance.

Description

Application of wheat TAALDHASE gene in regulation and control of wheat stem basal rot resistance

Technical Field

The invention relates to the technical field of molecular biology, in particular to application of wheat TAALDHASE gene in regulation and control of wheat stem rot resistance.

Background

Wheat is one of the most important food crops widely planted worldwide, and as a staple food for 40% of the population in the world, it plays an important role in food safety in various countries and in domestic life in China. The countries where wheat is mainly planted worldwide are china, united states, australia, canada, india, brazil, russia and the like. It has been reported that more than 200 wheat diseases have been found, and the most fungal disease species account for about 50% of the wheat diseases, and the losses caused by them can be more than 75%.

The stem basal rot is a serious disease which mainly damages the stem basal part of wheat and damages the ear part, and commonly occurs in the wheat producing area of the world to influence the yield and quality of wheat seeds. Because of the lack of disease-resistant varieties, chemical agents are mainly used for preventing the disease from being popular in production, and the efficiency is low and the environment is not protected. The wheat stem rot is widely generated and seriously damaged, and the currently reported control methods are various and comprise chemical seed dressing, reasonable fertilization, rotation, planting disease-resistant varieties and the like, but the methods cannot efficiently control the occurrence and progress of diseases. In order to reduce the influence of the plant diseases, the plant diseases are treated comprehensively under the condition of multiple pipes in actual production so as to reduce the loss caused by the plant diseases to the maximum extent. The method for controlling the wheat stem basal rot by planting the disease-resistant variety is an economical and safe control method which is currently effective. Because the wheat stem basal rot is controlled by multiple genes, the genetic efficiency is higher, and the resistance gene can be transferred into a background material with excellent agronomic characters through hybridization breeding or polymerization breeding, so that the resistance breeding aiming at the stem basal rot is realized.

The popularization of disease-resistant varieties is the most effective and environment-friendly method for preventing disease epidemics. Therefore, screening of disease-resistant germplasm, researching of pathogenicity of main pathogens of stem basal rot to wheat and disease-resistant mechanism of wheat to pathogens are beneficial to breeding popularization of disease-resistant varieties and establishment of new disease control methods.

Disclosure of Invention

The invention aims to provide an application of wheat TAALDHASE gene in regulating and controlling wheat stem basal rot resistance.

In a first aspect, the invention claims the use of TAALDHASE protein for modulating wheat stem basal rot resistance;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

The regulation is as follows: the expression level of TAALDHASE protein in wheat is reduced, and the resistance of the wheat to the stem rot is improved.

In this respect, this application is also within the scope of the invention if the wheat has an increased resistance to stalk rot because of the reduced activity of the TAALDHASE protein in wheat.

In (A2), the N-terminal and/or C-terminal linkage tag of the protein defined in (A1) is usually used for the convenience of purification or detection. The labels include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein, and the like. The following is the same.

Furthermore, the TAALDHASE protein can also be protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, has the same function and is derived from wheat; or a protein having the same function and having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in (A1) above. The following is the same.

Wherein, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained. The following is the same.

The 95% or more homology may be at least 96%, 97%, 98% identical. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical. The following is the same.

In a second aspect, the invention claims the use of a substance capable of reducing the amount of expression of TAALDHASE protein in wheat to increase the resistance of wheat stem rot;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in (A1).

In this aspect, the use of a substance capable of reducing the activity of TAALDHASE protein in wheat to increase the resistance of wheat to stalk rot is also within the scope of the present invention.

The "substance capable of reducing the expression level of TAALDHASE protein in wheat" may be a substance which is regulated at the level of gene transcription, a substance which is regulated at the level of gene transcription (i.e., a substance which regulates splicing or processing of primary transcripts), a substance which regulates RNA transport (i.e., a substance which regulates mRNA transport from the nucleus to the cytoplasm), a substance which regulates translation, or a substance which regulates mRNA degradation. The following is the same.

In a specific embodiment of the present invention, the "substance capable of reducing the expression level of TAALDHASE protein in wheat" is a BSMV genome editing vector; the BSMV genome editing vector consists of a BSMV-alpha vector, a BSMV-beta vector and a BSMV gamma-TAALDHASE vector; the BSMV gamma-TAALDHASE vector carries a silencing fragment shown in SEQ ID No.3 for silencing the coding gene of the TAALDHASE protein. Specifically, the BSMV gamma-TAALDHASE vector is obtained by inserting a DNA fragment shown in SEQ ID No.3 into the NheI cleavage site of the BSMV-gamma vector.

In a third aspect, the invention claims the use of a substance capable of reducing the amount of expression of TAALDHASE protein in wheat in any of the following:

(I) Reducing the disease index of the wheat stem basal rot;

(II) reducing the content of Deoxynivalenol (DON) in wheat.

In this respect, the use of substances capable of reducing the activity of TAALDHASE proteins in wheat in the above-mentioned (I) and (II) is also within the scope of the present invention.

The TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

The substance is a BSMV genome editing vector; the BSMV genome editing vector consists of a BSMV-alpha vector, a BSMV-beta vector and a BSMV gamma-TAALDHASE vector; the BSMV gamma-TAALDHASE vector carries a silencing fragment shown in SEQ ID No.3 for silencing the coding gene of the TAALDHASE protein. Specifically, the BSMV gamma-TAALDHASE vector is obtained by inserting a DNA fragment shown in SEQ ID No.3 into the NheI cleavage site of the BSMV-gamma vector.

In a fourth aspect, the invention claims a method of increasing the resistance of wheat to stalk rot.

The method for improving the resistance of wheat stem basal rot claimed by the invention can comprise the step of reducing the expression level of TAALDHASE protein in acceptor wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

In this aspect, the method may also include the step of reducing the activity of the TAALDHASE protein in the recipient wheat.

In a fifth aspect, the invention claims a method of reducing the index of the condition of stem basal rot in wheat and/or reducing the Deoxynivalenol (DON) content in wheat.

The method for reducing the disease index of the wheat stem basal rot and/or reducing the Deoxynivalenol (DON) content in the wheat, which is claimed by the invention, can comprise the step of reducing the expression level of TAALDHASE protein in the acceptor wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

Wherein the Deoxynivalenol (DON) is induced by Fusarium pseudograminearum.

In the fourth and fifth aspects of the foregoing, the method may be carried out by hybridization means or by transgenic means.

In a sixth aspect, the invention claims a method of breeding transgenic wheat with increased resistance to stalk rot.

The method for cultivating transgenic wheat with improved stem rot resistance, which is claimed by the invention, can comprise the following steps: inhibiting expression of nucleic acid molecules capable of expressing TAALDHASE protein in acceptor wheat to obtain transgenic wheat; the transgenic wheat has increased resistance to stalk rot as compared to the recipient wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

In a seventh aspect, the invention claims a method of breeding transgenic wheat with reduced index of stem basal rot disease and/or reduced in vivo Deoxynivalenol (DON) content.

The method for cultivating transgenic wheat with reduced stem basal rot disease index and/or reduced in vivo Deoxynivalenol (DON) content, which is claimed by the invention, can comprise the following steps: inhibiting expression of nucleic acid molecules capable of expressing TAALDHASE protein in acceptor wheat to obtain transgenic wheat; the transgenic wheat has a reduced disease index of said stem basal rot and/or a reduced in vivo Deoxynivalenol (DON) content compared to the recipient wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

Wherein the Deoxynivalenol (DON) is induced by Fusarium pseudograminearum.

In the sixth and seventh aspects of the foregoing, the inhibition of expression of the nucleic acid molecule capable of expressing the TAALDHASE protein in the recipient wheat may be achieved by any means that achieves this objective. In a specific embodiment of the invention, this is achieved in particular by introducing a BSMV genome editing vector into said recipient wheat; the BSMV genome editing vector consists of a BSMV-alpha vector, a BSMV-beta vector and a BSMV gamma-TAALDHASE vector; the BSMV gamma-TAALDHASE vector carries a silencing fragment shown in SEQ ID No.3 for silencing the coding gene of the TAALDHASE protein. Specifically, the BSMV gamma-TAALDHASE vector is obtained by inserting a DNA fragment shown in SEQ ID No.3 into the NheI cleavage site of the BSMV-gamma vector.

In the sixth and seventh aspects of the foregoing, the transgenic wheat is understood to include not only the first to second generation transgenic wheat, but also the progeny thereof. The transgenic wheat includes seeds, calli, whole plants and cells.

In the sixth and seventh aspects of the foregoing, the nucleic acid molecule capable of expressing the TAALDHASE protein is genomic DNA or mRNA.

In the sixth and seventh aspects of the foregoing, the nucleic acid molecule capable of expressing the TAALDHASE protein may be a DNA molecule as set forth in SEQ ID No. 2.

In the above aspects, the pathogenic bacteria of the stem base rot is fusarium pseudograminearum. In a specific embodiment of the invention, the fusarium pseudograminearum is specifically fusarium pseudograminearum CS3096.

In a specific embodiment of the invention, the wheat is wheat variety Pubing Zi 300.

In the above aspects, the application range of the related technical scheme is from the extension of wheat to the extension of gramineae or wheat genus or monocotyledonous plants or plants, which are all the scope of the present invention.

The present invention provides a wheat plant with a silenced TAALDHASE gene by introducing TAALDHASE gene derived from wheat (Triticum aestivum l.) into recipient wheat (protopine 300). Experiments prove that compared with an unslotted acceptor wheat control, under the condition of inoculation of pathogenic bacteria (fusarium pseudograminearum), the brown spot at the stem base of a plant with a silenced TAALDHASE gene is obviously less than the acceptor wheat control, and the growth vigor is better than the acceptor wheat control; in addition, silencing TAALDHASE gene significantly reduced the wheat grade index and the DON toxin content significantly reduced compared to the recipient wheat control, indicating that silencing of TAALDHASE gene significantly improved wheat resistance to stalk rot. The invention has important significance and application value for cultivating new varieties of wheat with stem rot resistance.

Drawings

FIG. 1 is a graph showing TAALDHASE transcript levels in BSMV: gamma and BSMV: TAALDHASE silenced wheat plants. In the figure, batch I and Batch II are two separate batches.

FIG. 2 is an analysis of resistance to shoot basal rot for BSMV TAALDHASE silenced wheat plants and BSMV gamma control wheat plants. A is the symptom of slight chlorosis of wheat leaves after inoculation of BSMV gamma or BSMV TAALDHASE. B is the disease phenotype of the fusarium pseudograminearum infecting the stems. Photographing after 14 days of inoculation of pathogenic bacteria.

FIG. 3 is a data analysis of BSMV TAALDHASE silenced wheat plants and BSMV gamma control wheat plants after infection with stem basal rot pathogen. A is the content of TAALDHASE transcript levels in leaves of BSMV: TAALDHASE silenced wheat plants and BSMV: gamma control wheat plants at 0, 12, 24, 48, 72 hpi (hpi represents hours after infection) under Fusarium pseudograminearum treatment. B is the disease index of wheat plants with BSMV being TAALDHASE silenced and BSMV being gamma control wheat plants. C is the content of Deoxynivalenol (DON) of mycotoxin induced by Fusarium pseudograminearum in the stem base of wheat plants with BSMV: TAALDHASE silencing and BSMV: gamma control wheat plants.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The wheat variety "Pubing Yi 300" in the following examples is provided by the professor Li Lihui of the institute of crop science, national academy of agricultural sciences. The above biological materials are publicly available from the applicant, and the obtained biological materials are used only for repeated experiments of the present invention and are not used for other purposes.

BSMV viral vector 3 component (BSMV- α, BSMV- β and BSMV- γ plasmids) in the following examples: the biological material is provided by Zhang Zengyan subject group of China institute of agriculture, academy of sciences of crops, and is recorded in ' Zhao Dan, zhao Jirong, huang Qian, li Ning, liu Yan, huang Zhanjing and Zhang Zengyan ' the yellow dwarf resistance of wheat TNBL1 gene is rapidly analyzed by using BSMV-VIGS technology, the biological material is obtained from applicant by the public in the paper of crop school 2011 (11) ' and is only used for experiments of the repeated invention, but not used for other purposes.

The BSMV in the examples described below is described in "Wanglong et al, cloning of the wheat SBE IIb gene and construction of the VIGS vector in tandem with the PDS gene, university of stone river, nature science edition, university journal, 2014-02-06", which is incorporated herein by reference, the public may obtain the above-mentioned biological materials from the applicant, and the above-mentioned biological materials obtained were used only for repeated experiments of the present invention and may not be used for other purposes.

Fusarium pseudograminearum (Fusarium pseudograminearum) CS3096 in the following examples: the public, as described in document "Jiang Y, Habib A, et al. Development of tightly linked markers and identification of candidate genes for Fusarium crown rot resistance in barley by exploiting a near-isogenic line-derived population. Theor Appl Genet. 2019 Jan;132(1):217-225. doi: 10.1007/s00122-018-3209-0. Epub 2018 Oct 16. PMID: 30327844.", can obtain the above-mentioned biological material from the applicant, and the obtained biological material is used only for repeated experiments of the present invention and is not used for other purposes.

PEASY-Blunt Blunt end cloning vector (TransGen Biotech, CB 101-01) and Trans1-T1 E.coli competence (TransGen Biotech, CD 501-02): the method is mainly used for gene cloning and sequencing.

The following examples were run using GRAPHPAD PRISM statistical software and the experimental results were expressed as mean ± standard deviation using Two-way ANOVA test, P < 0.05 (x) indicated significant differences and P < 0.01 (x) indicated very significant differences.

Example 1, TAALDHASE protein and the acquisition of the Gene encoding it

The primer is designed by PRIMER PREMIER 5 with the gene sequence of China spring TAALDHASE (TraesCS B03G 0823100) published by Ensembl website as reference, and the primer sequence is as follows:

TaALDHase-F：5'-TCTGTCTCC-ATGGGAAGCCTG-3'；

TaALDHase-R：5'-GTTGTTTCTTCCATCATCTC-3'。

And the specificity of the primers was verified by NCBI and WheatOmics websites, then gene cloning was performed using cDNA of ordinary wheat Pubing material 300 (stored in the national academy of agricultural science germplasm resource pool, provided by the professor Li Lihui of the national academy of agricultural science), and the cloned fragment was connected to an intermediate cloning vector pEASY-Blunt, and the TAALDHASE gene sequence was verified by sequencing.

Wheat seedlings are grown for about 7 days under normal conditions, and the leaves are taken and quickly frozen by liquid nitrogen and stored at the temperature of minus 80 ℃ for standby.

The total RNA of the wheat leaves is extracted by adopting a rapid extraction kit (ZOMANBIO, ZP 405-1) for total RNA of the plants, and cDNA is synthesized by removing gDNA and synthesizing cDNA mixed solution (AT 311-02) of the full gold company in one step. The PCR products were detected by electrophoresis on a 1.0% agarose gel.

The DNA fragment comprising SEQ ID No.2 was obtained by the PCR method. SEQ ID No.2 is TAALDHASE gene coding frame, coding protein shown in SEQ ID No. 1. The protein shown in SEQ ID No.1 is TAALDHASE protein.

Example 2 acquisition of wheat with VIGS silencing TAALDHASE Gene

1. Construction of recombinant VIGS vectors

1. Extracting total RNA of the common ice plant 300 wheat leaves, and carrying out reverse transcription to obtain cDNA.

2. The SGN VIGS tool website was used to design the optimal silencing fragment with reference to the Chinese spring TAALDHASE gene sequence published by Ensembl website (TraesCS B03G 0823100). Then, the cDNA in the step 1 is used as a template, specific primers are designed at two ends of the TAALDHASE gene optimal silencing fragment to amplify the gene, and PCR products are recovered. The amplification primers were as follows:

BSMV-TaALDHase-F：5'-TTGTCACATCGATGCCGAAAC-3'；

BSMV-TaALDHase-R：5'-TAGAAGAATGTAGTTCGTATTG-3'。

3. The PCR product amplified and recovered as described above was ligated with pEASY-Blunt vector (full gold, beijing) and transformed into E.coli Trans1-T1 competent cells (TransGen Biotech, CD 501-02) and plated on solid LB medium plates containing 50. Mu.g/L kanamycin, and cultured overnight at 37 ℃. And (3) performing bacterial liquid PCR detection on the clone colony of the escherichia coli, sequencing positive clones by a company, and preserving bacterial liquid with correct sequencing to obtain the pEASY-Blunt-TAALDHASE plasmid.

4. And (3) taking the plasmid obtained in the step (3) as a template, adopting a primer pair consisting of gamma-TAALDHASE-F and gamma-TAALDHASE-R to carry out PCR amplification to obtain a PCR amplification product, and carrying out gel recovery of the PCR amplification product.

γ-TaALDHase-F：5'-GATTCTTCTTCCGTTGCTAGC-TTGTCACATCGATGCCGAAAC-3'；

γ-TaALDHase-R：5'-TTTTTTTTTTTTTTAGCTAGC-TAGAAGAATGTAGTTCGTATTG-3'。

The underlined portion is the nhei recognition sequence.

5. The vector BSMV-gamma is digested with restriction enzyme NheI, and the vector backbone is recovered.

6. The PCR product of step 4 and the vector backbone of step 5 were ligated using the In-Fusion technique of Takara, inc., to obtain recombinant plasmid BSMV gamma-TAALDHASE. Based on the sequencing results, the recombinant plasmid BSMV gamma-TAALDHASE was structurally described as follows: a recombinant vector of the DNA fragment shown in SEQ ID No.3 is inserted into the NheI cleavage site of the vector BSMV-gamma.

2. TAALDHASE Gene silencing wheat acquisition

1. Linearization of VIGS vectors:

the vector BSMV-alpha is digested with restriction enzyme MluI, and the vector backbone is recovered.

The vector BSMV-. Beta.was digested with the restriction enzyme SpeI, and the vector backbone was recovered.

The vector BSMV-gamma is digested with restriction enzyme NheI, and the vector backbone is recovered.

The vector BSMV: gamma-PDS was digested with restriction enzyme BssHII, and the vector backbone was recovered.

The vector BSMV gamma-TAALDHASE was digested with restriction enzyme MluI, and the vector backbone was recovered.

2. After completion of step 1, each recovered vector was subjected to in vitro transcription according to the RiboMAX ™ LARGE SCALE RNA Production Systems-T7 kit from Promega corporation.

3. Preparation of FES buffer

Mother liquor 5× GPbuffer: glycine 1.877 g, dipotassium hydrogen phosphate 2.613 g, RNase-Free ddH ₂ O to 50mL, sterilizing 20min in a sterilizing pot for later use.

1 XGP Buffer, sodium pyrophosphate 5g, bentonite 5g, diatomite 5g, RNase-Free ddH ₂ O of FES Buffer 100 mL were prepared to a constant volume of 500 mL, and sterilized in a sterilizing pot of 20 min for use.

Friction fluid composition for different treatments: taking 2.5 mu L of products of in-vitro transcription, namely alpha, beta, gamma/gamma-PDS/gamma-TAALDHASE, according to the following steps of 1:1:1 (volume ratio), and diluting with RNase-Free ddH ₂ O, sucking 5 μl of the diluted mixture, and mixing with 90 μl of FES buffer to obtain FES mixture. This experiment sets up 4 groups at every time, is respectively: complete control (WT), viral control (α+β+γ), albino positive control (α+β+γ -PDS) and gene silencing group (α+β+γ -TAALDHASE).

4. After the step 3 is completed, a small amount of RNase-Free ddH ₂ O is sprayed on the surface of wheat leaves (common ice resource 300) to be infected, 8-10 mu L of FES buffer solution is sprayed on clean gloves, 3 times of friction are carried out on second leaves of seedlings, the force is controlled during friction, a small amount of RNase-Free ddH ₂ O is sprayed from top to bottom after friction is completed, and the operation of changing the clean gloves is needed for each treatment. After virus inoculation, the virus is placed in a 23+/-2 ℃ incubator for heat preservation, moisture preservation and light shielding for 24h, and then is adjusted to 16h/8h of light-dark period for culture, and the phenotype change is observed and recorded regularly.

5. After the step 4 is completed, after the gamma-PDS positive control phenotype appears, the gene silencing efficiency of the wheat plants is measured. Taking wheat leaves infected by 'common ice gene 300' and BSMV virus, extracting total RNA and carrying out reverse transcription to obtain cDNA, carrying out qRT-PCR by using a cDNA template, adopting an action gene as an internal reference gene, and detecting the relative expression quantity of TAALDHASE genes.

The primers used to detect TAALDHASE genes were as follows:

RT-TaALDHase-F：5'-TGGCTCGGATCTTGAACACA-3'；

RT-TaALDHase-R：5'-GAGCAGTGGGCCGAATATCT-3'。

The primers used for detecting the action gene are as follows:

RT-Actin-F：5'-CCTCTCTGCGCCAATCGT-3'；

RT-Actin-R：5'-TCAGCCGAGCGGGAAATTGT-3'。

The results are shown in FIG. 1. And detecting TAALDHASE gene relative expression quantity by qPCR, and selecting action as an internal reference gene. Each data point is the average of three replicates (±sd). And infected with BSMV: the transcript level of the wheat plant (corresponding to the "virus blank group (alpha+beta+gamma)") of BSMV TAALDHASE was significantly reduced compared with the wheat plant (corresponding to the "gene silencing group (alpha+beta+gamma-TAALDHASE)") of the above, indicating successful silencing of the TAALDHASE gene.

Example 3 analysis of sensitivity of wheat to Fusarium pseudograminearum

Wheat plants obtained in example 2 were inoculated with fusarium graminearum, fusarium graminearum CS3096, which was successfully silenced with TAALDHASE genes, were sampled 0-72 hours after infection for histological observation and RNA isolation, and the silencing efficiency of the genes was analyzed by real-time reverse transcription PCR (qRT-PCR) (see example 2 for specific methods). The stem base was sampled 14 days after infection for analysis of Deoxynivalenol (DON) yield, while the status of infection to the plants was photographed 14 days after infection.

The inoculation method comprises the following steps:

1. preparation of a culture medium:

Peeling fresh potato, cutting into small pieces, weighing 200 g pieces of potato, placing into a pot, and boiling with distilled water. After about 20 min, the potato pieces were filtered with gauze as they could be easily mashed with a glass rod. Reheating the filtrate, sequentially adding agar powder 15 g and glucose 20g into a pot, stirring uniformly, fixing volume in a 1L beaker, packaging after slightly cooling, packaging into small-capacity conical bottles, and performing wet heat sterilization again under the condition of 0.1 Mpa at 121 ℃ for 20 min for later use. Selecting millet with uniform size, boiling in boiling water for 3 min, pouring out, washing with cold water, spreading on clean gauze, airing at ventilation position until no water is present on the surface, airing, packaging into 150 mL triangular flask, sealing bottle mouth, sterilizing at 121deg.C under 0.1 Mpa, and sterilizing under damp-heat for 30 min.

2. And (5) propagation of strains:

The healthy and vigorous fusarium pseudograminearum CS3096 bacterial blocks with hyphae are picked from the edge of a PDA culture plate of the new activated bacteria, are inoculated into a sterilized millet culture medium (prepared in the step 1) and are cultured for 7 days at the temperature of 25 ℃, and are shaken for 2 times per day to uniformly distribute pathogenic bacteria for material inoculation.

3. Wheat plant inoculation

The 7 cm ×7 cm square boxes for wheat (TAALDHASE gene silencing successful wheat plants obtained in example 2) were inoculated with 0.4 g germ-bearing millet (prepared in step 2), the boxes were evenly spread (spread on the upper layer of the soil matrix) and one grain of the wheat fungus was ensured at the base of each wheat seedling, and the culture conditions were the same as in step 2, and watered once every 3 days. The experiment was performed with BSMV gamma control wheat and 300 wheat plants of plain ice without any treatment.

The results are shown in fig. 2 and 3. Figure 2 shows that the BSMV TAALDHASE silenced wheat plants exhibited significantly less stem browning than control wheat plants after Fusarium pseudograminearum infection. This indicates that wheat plants with silenced TAALDHASE gene are more resistant to stem basal rot. FIG. 3A shows that BSMV TAALDHASE-induced TAALDHASE gene transcript in wheat leaves was reduced by 48% -68% at 0, 12, 24, 48, 72 hpi under Fusarium pseudograminearum treatment. FIG. 3B shows that BSMV TAALDHASE-induced wheat plant disease index is lower than BSMV-gamma control plants. FIG. 3C shows that Fusarium pseudograminearum-induced mycotoxin Deoxynivalenol (DON) content in the shoot base of wheat plants induced with BSMV TAALDHASE was lower than that of BSMV: gamma control plants. Each data point in fig. 3 is the average of three replicates (±sd). This indicates that wheat plants with silenced TAALDHASE gene have lower disease grade index and lower DON content.

The above results indicate that the silencing of TAALDHASE gene can enhance wheat resistance to wheat stem basal rot. This means that TAALDHASE gene down regulates wheat resistance to wheat stem basal rot.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. Use of TAALDHASE protein in regulating and controlling wheat stem rot resistance;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

The regulation is as follows: the expression quantity of TAALDHASE protein in wheat is reduced, and the resistance of the wheat to the stem rot is improved;

the pathogenic bacteria of the stem basal rot are Fusarium pseudograminearum.

2. The application of a substance capable of reducing the expression quantity of TAALDHASE protein in wheat in improving the stem rot resistance of wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

The substance is a BSMV genome editing vector; the BSMV genome editing vector consists of a BSMV-alpha vector, a BSMV-beta vector and a BSMV gamma-TAALDHASE vector; the BSMV gamma-TAALDHASE vector carries a silencing fragment shown in SEQ ID No.3 for silencing the coding gene of the TAALDHASE protein;

the pathogenic bacteria of the stem basal rot are Fusarium pseudograminearum.

3. Use of a substance capable of reducing the expression level of TAALDHASE protein in wheat in any of the following:

(I) Reducing the disease index of the wheat stem basal rot;

(II) reducing the content of deoxynivalenol in wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

the pathogenic bacteria of the stem basal rot are Fusarium pseudograminearum.

4. A method for increasing the resistance of wheat stem basal rot comprising the step of reducing the expression level of TAALDHASE protein in recipient wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

the pathogenic bacteria of the stem-based rot are Fusarium pseudograminearum;

Reducing the amount of expression of the TAALDHASE protein in the recipient wheat by introducing a BSMV genome editing vector into the recipient wheat; the BSMV genome editing vector consists of a BSMV-alpha vector, a BSMV-beta vector and a BSMV gamma-TAALDHASE vector; the BSMV gamma-TAALDHASE vector carries a silencing fragment shown in SEQ ID No.3 for silencing the coding gene of the TAALDHASE protein.

5. A method for reducing the index of the condition of stem-based rot of wheat and/or reducing the deoxynivalenol content in wheat, comprising the step of reducing the expression level of TAALDHASE protein in recipient wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

the pathogenic bacteria of the stem-based rot are Fusarium pseudograminearum;

6. A method of breeding transgenic wheat with increased resistance to stalk rot comprising the steps of: inhibiting expression of nucleic acid molecules capable of expressing TAALDHASE protein in acceptor wheat to obtain transgenic wheat; the transgenic wheat has increased resistance to stalk rot as compared to the recipient wheat;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

the pathogenic bacteria of the stem-based rot are Fusarium pseudograminearum;

Inhibiting expression of a nucleic acid molecule capable of expressing the TAALDHASE protein in the recipient wheat is achieved by introducing a BSMV genome editing vector into the recipient wheat; the BSMV genome editing vector consists of a BSMV-alpha vector, a BSMV-beta vector and a BSMV gamma-TAALDHASE vector; the BSMV gamma-TAALDHASE vector carries a silencing fragment shown in SEQ ID No.3 for silencing the coding gene of the TAALDHASE protein.

7. A method for breeding transgenic wheat with reduced stem basal rot disease index and/or reduced in vivo deoxynivalenol content, comprising the steps of: inhibiting expression of nucleic acid molecules capable of expressing TAALDHASE protein in acceptor wheat to obtain transgenic wheat; compared with the receptor wheat, the transgenic wheat has reduced stem basal rot disease index and/or reduced in vivo deoxynivalenol content;

the TAALDHASE protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

the pathogenic bacteria of the stem-based rot are Fusarium pseudograminearum;

8. The method according to claim 6 or 7, characterized in that: the nucleic acid molecule capable of expressing TAALDHASE protein is the DNA molecule shown as SEQ ID No. 2.